COVALENT LINKAGE OF STREPTOKINASE TO RECOMBINANT HIRUDIN - A NOVEL THROMBOLYTIC AGENT WITH ANTITHROMBOTIC PROPERTIES

In a continuing effort to create an agent which has both thrombolytic and antithrombotic properties, streptokinase (SEC) was covalently boun d to the potent antithrombin agent recombinant hirudin (rHir). Linkage of SK to I-125-rHir was accomplished via heterobifunctional crosslink ers in an average molar ratio of 1:1. The I-125-rHir-SK complex was pu rified from starting components by anion exchange and gel filtration c hromatography. The major band containing covalently bound I-125-rHir h ad a molecular weight of 53 kDa as determined by SDS-PAGE and autoradi ography. Biologic activity of each component was then assayed utilizin g the chromogenic substrate for each compound. Complex bound I-125-rHi r exhibited a 1.2 fold decrease in thrombin inhibition when compared t o concentrations of I-125-rHir greater than 3.13 nM. Complex bound I-1 25-SK, replacing the I-125 label on rHir. displayed a 7.9-fold loss in plasminogen activation when compared to I-125-SK. These chromogenic a ssay results were not adversely altered in the presence of the convers e compound's substrate. The I-125-SK-rHir complex (examined at Various concentrations) also demonstrated a 0.17- to 17-fold greater affinity for thrombin immobilized onto Sepharose beads as compared to I-125-SK . These findings indicate the rHir-SK complex maintained both thrombol ytic and antithrombin properties while also obtaining affinity for imm obilized thrombin.